STUDIES ON THE METABOLISM OF AUTOTROPHIC BACTERIA : II. THE NATURE OF THE CHEMOSYNTHETIC REACTION

In a study of chemosynthesis (the fixation of CO₂ by autotrophic bacteria in the dark) in Thiobacillus thiooxidans, the data obtained support the following conclusions: 1. CO₂ can be fixed by "resting cells" of Thiobacillus thiooxidans; the fixation is not "growth bound." 2. The physiological condition of the cell is of considerable importance in determining CO₂ fixation. 3. CO₂ fixation can occur in the absence of oxidizable sulfur in "young" cells. The extent of this fixation appears to be dependent upon the pCO₂. 4. CO₂ fixation can also occur under anaerobic conditions and the presence of sulfur does not influence such fixation. 5. However, in the CO₂ fixation by cells in the absence of sulfur, only a limited amount of CO₂ can be fixed. This amount is approximately 40 µl. CO₂ per 100 micrograms bacterial nitrogen. After a culture has utilized this amount of CO₂ it no longer has the ability to fix CO₂ but releases it during its respiration. 6. Relatively short periods of sulfur oxidation can restore the ability of cells to fix CO₂ under conditions where sulfur oxidation is prevented. 7. It is possible to oxidize sulfur in the absence of CO₂ and to store the energy thus formed within the cell. It is then possible to use this energy at a later time for the fixation of CO₂ in the entire absence of sulfur oxidation. 8. Cultures of Thiobacillus thiooxidans respiring on sulfur utilize CO₂ in a reaction which proceeds to a zero concentration of CO₂ in the atmosphere. 9. CO₂ may act as an oxidizing agent for sulfur. 10. Hydrogen is not utilized by the organism. 11. It is possible to selectively inhibit sulfur oxidation and CO₂ fixation.     

ON THE GROWTH AND RESPIRATION OF SULFUR-OXIDIZING BACTERIA

1. It is shown that Sulfomonas thiooxidans oxidizes elementary sulfur completely to sulfuric acid. Sodium thiosulfate is oxidized by this organism completely to sulfate. Sulfomonas thiooxidans differs, in this respect, from various other sulfur-oxidizing bacilli which either produce elementary sulfur, from the thiosulfate, or convert it into sulfates and persulfates. 2. The organism derives its carbon from the CO₂ of the atmosphere, but is incapable of deriving the carbon from carbonates or organic matter. 3. The S:C, or ratio between the amount of sulfur oxidized to sulfate and amount of carbon assimilated chemosynthetically from the CO₂ of the atmosphere, is, with elementary sulfur as a source of energy, 31.8, and with thiosulfate 64.2. The higher ratio in the case of the thiosulfate is due to the smaller amount of energy liberated in the oxidation of sulfur compound than in the elementary form. 4. Of the total energy made available in the oxidation of the sulfur to sulfuric acid, only 6.65 per cent is used by the organism for the reduction of atmospheric CO₂ and assimilation of carbon. 5. Sulfates do not exert any injurious effect upon sulfur oxidation by Sulfomonas thiooxidans. Any effect obtained is due to the cation rather than the sulfate radical. Nitrates exert a distinctly injurious action both on the growth and respiration of the organism. 6. There is a definite correlation between the amount of sulfur present and velocity of oxidation, very similar to that found in the growth of yeasts and nitrifying bacteria. Oxidation reaches a maximum with about 25 gm. of sulfur added to 100 cc. of medium. However, larger amounts of sulfur have no injurious effect. 7. Dextrose does not exert any appreciable injurious effect in concentrations less than 5 per cent. The injurious effect of peptone sets in at 0.1 per cent concentration and brings sulfur oxidation almost to a standstill in 1 per cent concentration. Dextrose does not exert any appreciable influence upon sulfur oxidation and carbon assimilation from the carbon dioxide of the atmosphere. 8. Sulfomonas thiooxidans can withstand large concentrations of sulfuric acid. The oxidation of sulfur is affected only to a small extent even by 0.25 molar initial concentration of the acid. In 0.5 molar solutions, the injurious effect becomes marked. The organism may produce as much as 1.5 molar acid, without being destroyed. 9. Growth is at an optimum at a hydrogen ion concentration equivalent to pH 2.0 to 5.5, dropping down rapidly on the alkaline side, but not to such an extent on the acid, particularly when a pure culture is employed. 10. Respiration of the sulfur-oxidizing bacteria can be studied by using the filtrate of a vigorously growing culture, to which a definite amount of sulfur is added, and incubating for 12 to 24 hours.     

THE PRESENCE OF AN ENDOGENOUS RESPIRATION IN THE AUTOTROPHIC BACTERIA

It is shown that there exists in the autotrophic bacterium Thiobacillus thiooxidans a measurable oxygen uptake in the absence of the specific nutrient (sulfur). This respiration is shown to be due to the utilization of organic materials which must have been previously synthesized by the chemosynthetic process, providing evidence that autotrophic bacteria contain a dissimilatory process which involves the breakdown of organic materials and furnishes energy for the maintenance of the cell during periods in which the specific nutrient is absent. This is entirely in accord with the work of Bömeke (1939), who provided similar types of proof for Nitrosomonas and Nitrobacter. One may conclude, therefore, that autotrophic bacteria possess an endogenous respiration which involves the utilization of previously synthesized organic materials.     

STUDIES ON THE METABOLISM OF THE AUTOTROPHIC BACTERIA : III. THE NATURE OF THE ENERGY STORAGE MATERIAL ACTIVE IN THE CHEMOSYNTHETIC PROCESS

In the autotrophic bacterium, Thiobacillus thiooxidans, the oxidation of sulfur is coupled to transfers of phosphate from the medium to the cells. CO₂ fixation is coupled to transfers of inorganic phosphate from the cells to the medium and is dependent, in the absence of concomitant sulfur oxidation, upon the amount of phosphate previously taken up during sulfur oxidation. The energy reservoir, which is formed by sulfur oxidation in the absence of CO₂ and which can be released for the fixation of CO₂ under conditions which do not permit sulfur oxidation, is a phosphorylated compound and the data suggest that the energy is stored in the cell as phosphate bond energy. It is possible to oxidize sulfur at a constant rate for hours in the absence of CO₂. The phosphate energy formed during this process is probably released by cell phosphotases. It is possible to inhibit these phosphotases by means of inorganic phosphate and thus to inhibit sulfur oxidation in the absence of CO₂. In the presence of CO₂, where alternative uses for the phosphate energy are available, the inhibition is relieved. Sulfur oxidation (energy input) is coupled, not to CO₂ fixation, but to phosphate esterification. CO₂ fixation (energy utilization) is coupled with phosphate release.     

STUDIES ON THE METABOLISM OF A SULFUR-OXIDIZING BACTERIUM: I. OXIDATION OF SULFUR

1. Thiobacillus thiooxidans, isolated in our laboratory, was foundto oxidize sulfur, but not thiosulfate. Tetrathionate is alsooxidized slightly. Its ability to oxidize sulfur is inactivatedeven by such a mild treatment as keeping the cells in a frozenstate.
2. Inhibitory action of alcohols on the sulfur oxidationincreasesas the length of carbon chain of alcohols increases.Carboxylicacids do not inhibit the sulfur oxidation at pH abovetheirpK, while they strongly inhibit the reaction at pH belowthepK.
3. The sulfur oxidation is inhibited by cyanide, azide,diethyldithiocarbamateand carbon monoxide, and the inhibitionby carbon monoxide isnot reversed by light. These results suggestthe presence ofmetal enzymes in the sulfur oxidation system.The terminal enzymeof this reaction appears to be differentfrom the usual cytochromeoxidase.

(Received May 13, 1960; )     

Studies on transfer processes in mixing vessels: effect of particles on gas-liquid mass transfer using modified Rushton turbine agitators

The influence of carbon dioxide concentration in liquid medium on elemental sulphur oxidation by Thiobacillus thiooxidans bacteria presented in this paper can be divided into 3 differing relationships. First relationship shows increase of sulphur biooxidation rate with increase of carbon dioxide concentration in liquid medium. Second one shows decrease of S⁰ oxidation rate with increase of CO₂ concentration in nutrient and in the third relationship there is no influence of carbon dioxide concentration on sulphur oxidation by Thiobacillus thiooxidans bacteria. The influence of carbon dioxide concentration in liquid nutrient on alive bacteria concentration in liquid medium is similar to those described above.     

Oxidation of Elemental Sulfur to Sulfite by Thiobacillus thiooxidans Cells

Thiobacillus thiooxidans cells oxidized elemental sulfur to sulfite, with 1 mol of O₂ consumption per mol of sulfur oxidized to sulfite, when the oxidation of sulfite was inhibited with 2-n-heptyl-4-hydroxyquinoline N-oxide.     

STUDIES ON THE METABOLISM OF A SULFUR-OXIDIZING BACTERIUM III. POSTOXIDATIVE FIXATION OF CARBON DIOXIDE

1. The cells of Thiobacillus thiooxidans, which had been in contactwith sulfur or sulfide in air (or CO₂-free air), could fix addedCOa very rapidly after replacing air with nitrogen. This fixationis designated as the postoxidative fixation.
2. "Preoxidation"of the sulfur compounds is mandatory for theoccurrence of thepostoxidative fixation.
3. The cells which had preliminarilyoxidized sulfide could notshow the CO₂-fixation, when theywere placed under an anaerobiccondition in the absence of thesulfur compound.
4. These results indicate that sulfur compoundsmay have an importantrole as the electron donor for the reductionof CO₂, besidestheir role as the substrate of respiration tosecure energyfor the fixation of CO₂

(Received March 6, 1962; )     

Studies on the metabolism of a sulfur-oxidizing bacterium IV.1 Growth and oxidation of sulfur compounds in Thiobacillus thiooxidans

By immersing a few small cellophane bags containing BaCO³ powderin STARKEY's medium, the duration of lag phase in the growthof Thiobacillus thiooxidans is minimized and the yield of cellsis increased ten times that of the previous method. The activitiesof oxidation for sulfur and sulfite change with growth. Sulfiteis oxidized at a comparable rate to that of sulfur oxidationat pH values between 6.0 and 6.5. In the presence of cysteineor glutathione, thiosulfate can be oxidized at a pH above 5.0.At pH values below 4.5, apparent oxidation of thiosulfate andtetrathionate to sulfate is observed. This result is accountedfor by the facts that thiosulfate is decomposed to sulfur andsulfite under the acidic condition at pH values below 4.5, andthat tetrathionate is reduced to thiosulfate enzymatically.In the oxidation of tetrathionate, oxygen uptake begins aftera lag phase, the duration of which depends on the concentrationsof cells and of tetrathionate. Cysteine is oxidized to cystine.The oxidation is strongly inhibited by metal-chelating agents.The cysteine oxidizing activity is, however, quite stable andis not lost by treating cells with organic solvents, sonic oscillation,by heating or lyophilization. ¹III=References (11). ²Partly supported by a grant from the Ministry of Education.     

Effect of Various Ions, pH, and Osmotic Pressure on Oxidation of Elemental Sulfur by Thiobacillus thiooxidans

The oxidation of elemental sulfur by Thiobacillus thiooxidans was studied at pH 2.3, 4.5, and 7.0 in the presence of different concentrations of various anions (sulfate, phosphate, chloride, nitrate, and fluoride) and cations (potassium, sodium, lithium, rubidium, and cesium). The results agree with the expected response of this acidophilic bacterium to charge neutralization of colloids by ions, pH-dependent membrane permeability of ions, and osmotic pressure.     

Leaching of Pyrite by Acidophilic Heterotrophic Iron-Oxidizing Bacteria in Pure and Mixed Cultures

Seven strains of heterotrophic iron-oxidizing acidophilic bacteria were examined to determine their abilities to promote oxidative dissolution of pyrite (FeS₂) when they were grown in pure cultures and in mixed cultures with sulfur-oxidizing Thiobacillus spp. Only one of the isolates (strain T-24) oxidized pyrite when it was grown in pyrite-basal salts medium. However, when pyrite-containing cultures were supplemented with 0.02% (wt/vol) yeast extract, most of the isolates oxidized pyrite, and one (strain T-24) promoted rates of mineral dissolution similar to the rates observed with the iron-oxidizing autotroph Thiobacillus ferrooxidans. Pyrite oxidation by another isolate (strain T-21) occurred in cultures containing between 0.005 and 0.05% (wt/vol) yeast extract but was completely inhibited in cultures containing 0.5% yeast extract. Ferrous iron was also needed for mineral dissolution by the iron-oxidizing heterotrophs, indicating that these organisms oxidize pyrite via the “indirect” mechanism. Mixed cultures of three isolates (strains T-21, T-23, and T-24) and the sulfur-oxidizing autotroph Thiobacillus thiooxidans promoted pyrite dissolution; since neither strains T-21 and T-23 nor T. thiooxidans could oxidize this mineral in yeast extract-free media, this was a novel example of bacterial synergism. Mixed cultures of strains T-21 and T-23 and the sulfur-oxidizing mixotroph Thiobacillus acidophilus also oxidized pyrite but to a lesser extent than did mixed cultures containing T. thiooxidans. Pyrite leaching by strain T-23 grown in an organic compound-rich medium and incubated either shaken or unshaken was also assessed. The potential environmental significance of iron-oxidizing heterotrophs in accelerating pyrite oxidation is discussed.     

STUDIES ON THE METABOLISM OF A SULFUR-OXIDIZING BACTERIUM II. THE SYSTEM OF CO2-FIXATION IN THIOBAC1LLUS THIOOXIDANS

1. In the early stage of CO₂-fixation by Thiobacillus thiooxidans,which was incubated aerobically in the presence of sulfur, mostpart of the fixed carbon was found in the phosphate ester fraction.
2. The fixation was inhibited by NaF, picolinic acid, PCMB, azide,dipyridyl, o-phenanthroline, monoiodoacetic acid, and arsenite,each in the concentration range where the sulfur oxidation wasnot affected strongly.
3. The crude extract of this organismcould fix CO₂ in the presenceof ATP, R-5-P and Mg⁺⁺. Most partof the fixed carbon was foundin PGA.
4. The crude extract showedthe CO₂-fixation coupled with the H₂S-oxidationin the presenceof ADP.
5. An appreciable reduction of PGA could not be detectedin thepresence of reducing systems, involving TPNH and DPNH.

(Received March 6, 1962; )     

Photosynthetic activity and components of the electron transport chain in the aerobic bacteriochlorophyll <Emphasis Type="Italic">a</Emphasis>-containing bacterium <Emphasis Type="Italic">Roseinatronobacter thiooxidans</Emphasis>

Bioenergetics of the aerobic bacteriochlorophyll a-containing (BCl a) bacterium (ABC bacterium) Roseinatronobacter thiooxidans is a combination of photosynthesis, oxygen respiration, and oxidation of sulfur compounds under alkaliphilic conditions. The photosynthetic activity of Rna. thiooxidans cells was established by the photoinhibition of cell respiration and reversible photobleaching discoloration of the BCl a of reaction centers (RC), connected by the chain of electron transfer with cytochrome c ₅₅₁ oxidation. The species under study, like many purple bacteria and some of the known ABC bacteria, possesses a light-harvesting pigment-protein (LHI) complex with the average number of 30 molecules of antenna BCl a per one photosynthetic RC. Under microaerobic growth conditions, the cells contained bc ₁ complex and two terminal oxidases: cbb ₃-cytochrome oxidase and the alternative cytochrome oxidase of the a ₃ type. Besides, Rna. thiooxidans was shown to have several different soluble low- and high-potential cytochromes c, probably associated with the ability of utilizing sulfur compounds as additional electron donors.     

Pyrite oxidation in acid sulphate soils: The role of miroorganisms

Summary A study has been made of microbial processes in the oxidation of pyrite in aicd sulphate soil material. Such soils are formed during aeration of marine muds rich in pyrite (FeS₂). Bacteria of the type ofThiobacillus ferrooxidans are mainly responsible for the oxidation of pyrite, causing a pronounced acidification of the soil. However, becauseThiobacillus ferrooxidans functions optimally at pH values bellow 4.0, its activity cannot explain the initial pH drop from approximately neutral to about 4. This was shown to be a non-biological process, in which bacteria play an insignificant part. AlthoughThiobacillus thioparus andThiobacillus thiooxidans were isolated from the acidifying soil, they did not stimulate oxidation of FeS₂, but utilized reduced sulphur compounds, which are formed during the non-biological oxidation of FeS₂.Ethylene-oxide-sterilized and dry-sterilized soil inoculated with pure cultures of mixtures of various thiobacilli or with freshly sampled acid sulphate soil soil did not acidify faster than sterile blanks.Thiobacillus thiooxians. Thiobacillus thioparus. Thiobacillus intermedius andThiobacillus perometabolis increased from about 10⁴ to 10⁵ cells/ml in media with FeS₂ as energy source. However, FeS₂ oxidation in the inoculated media was not faster than in sterile blanks.Attempts to isolate microorganisms other thanThiobacillus ferrooxidans, like metallogenium orLeptospirillum ferrooxidans, which might also be involved in the oxidation of FeS₂ were not successful.Addition of CaCO₃ to the soil prevented acidification but did not stop non-biological oxidation of FeS₂.     

Bacterial leaching of metals from sewage sludge

Summary Thiobacillus thiooxidans is capable of oxidizing sulfur in digested sludge, while decreasing the pH value from about 5.5 to, say, 1.0 to 1.5. Insoluble metal sulfides can be solubilized through this acidification. Thiobacillus ferrooxidans oxidises pyritic ore in the presence of 6% centrifuged sludge if the pH value is adjusted to about 2.5. When mixing T. thiooxidans and T. ferrooxidans with sludge and 1% sulfur, the former acidifies the sludge and the latter oxidizes metal sulfides; together they solubilize more metal than T. thiooxidans alone. The following metals solubilized from their sulfides have been investigated so far: iron, copper, zinc, nickel, and cadmium. The possibility of recycling metals from sewage sludge with this method is discussed.     

THE EFFECT OF LOW PRESSURES ON CELL OXIDATION

1. A method is described for measuring tissue oxidation under reduced barometric pressure. 2. The oxygen uptake of yeast is diminished by low barometric pressures to a greater extent than by a reduction of the partial pressure of oxygen, to a corresponding degree, at atmospheric pressure. 3. This effect of low pressure is not observed with certain in vitro oxidation systems. 4. The anaerobic respiration (carbon dioxide production) of yeast is not at all affected by low pressures. 5. The inhibition of tissue oxidation caused by carbon monoxide is removed by lowering the pressure.     

Bacterial Leaching of Metal Sulfides Proceeds by Two Indirect Mechanisms via Thiosulfate or via Polysulfides and Sulfur

The acid-insoluble metal sulfides FeS₂, MoS₂, and WS₂ are chemically attacked by iron(III) hexahydrate ions, generating thiosulfate, which is oxidized to sulfuric acid. Other metal sulfides are attacked by iron(III) ions and by protons, resulting in the formation of elemental sulfur via intermediary polysulfides. Sulfur is biooxidized to sulfuric acid. This explains leaching of metal sulfides by Thiobacillus thiooxidans.     

Kinetics of sulfur oxidation by thiobacillus ferrooxidans

Abstract

Thiobacillus ferrooxidans ATCC 23270 was grown with elemental sulfur as the energy source. Substrate oxidation was measured using a Clark‐type oxygen electrode. Whole cells demonstrated a broad pH optimum for sulfur oxidation between pH 2.0 and 8.0. The V _max and K_sfor sulfur oxidation varied depending on pH. Sulfite was oxidized at 227 nmol O₂/min/mg protein. Thiosulfate oxidation was slow, and tetrathionate oxidation was not detected. At a concentration of 2 mM, sodium azide completely inhibited sulfur, sulfite, and thiosulfate oxidation. Inhibition by N‐ethylmaleimide, antimycin A, and 2‐heptyl‐4‐hydroxyquinoline N‐oxide varied with substrate.     

Fatty Acids of Thiobacillus thiooxidans

Fatty acid spectra were made on Thiobacillus thiooxidans cultures both in the presence and absence of organic compounds. Small additions of glucose or acetate had no significant effect either on growth or fatty acid content. The addition of biotin had no stimulatory effect but did result in slight quantitative changes in the fatty acid spectrum. The predominant fatty acid was a C₁₉ cyclopropane acid.     

Nickel Inhibition of the Growth of a Sulfur-oxidizing Bacterium Isolated from Corroded Concrete

A sulfur-oxidizing bacterium strain NB1-3 isolated from corroded concrete was a Gram negative, non-spore-forming, and rod-shaped bacterium (0.5–1.0x 1.5–2.0μm) with a polar flagellum. Strain NB1-3 had its optimum temperature and pH for growth at 30°C and 3.0–4.0, respectively. Strain NB1-3 had enzyme activities that oxidized elemental sulfur, thiosulfate, tetrathionate, and sulfide and the activity to incorporate ¹⁴CO₂ into the cells. The mean G+C content of the DNA was 52.9 mol%. These results indicate that strain NB1-3 is Thiobacillus thiooxidans. Since nickel has been known to protect concrete from corrosion, the effect of Ni on the growth of strain NB1-3 was studied. The cell growth on tiosulfate-, elemental sulfur-, or tetrathionate-medium was completely inhibited by 0.1% metal nickel or 5mM NiSO₄. Both cellular activities of elemental sulfur oxidation and CO₂ incorporation were strongly inhibited by 5mM NiSO₄. The amounts of Ni in cells with or without nickel treatment were 1.7 and 160.0 nmol/mg protein, respectively. These results indicate that nickel binds to strain NB1-3 cells and inhibits enzymes involved in sulfur oxidation of this bacterium, and as a result, inhibits cell growth.